ABSTRACT The goal of this project is to determine how dominant mutations in glycyl tRNA synthetase (GARS) cause peripheral axon degeneration in Charcot-Marie-Tooth disease (CMT) type 2D. Six tRNA synthetase genes are associated with CMT, but these are ubiquitous ?housekeeping genes? that charge amino acids onto their cognate tRNAs, and why they cause such a specific disease is unclear. A loss of tRNA charging activity would not explain the disease specificity and is not completely consistent with clinical genetics. Our work in mouse models of CMT2D strongly suggests that a toxic gain-of-function causes neuropathy. Mice heterozygous for amino acid substitutions in Gars (C201R and P278KY) develop peripheral neuropathy, whereas mice heterozygous for a null allele of Gars are unaffected, arguing against a haploinsufficiency. Also, 10-20 fold overexpression of transgenic wild-type GARS does not suppress the neuropathy, arguing against a loss of function. Through collaborative work, we now have two intriguing gain-of-function mechanisms. First, mutant forms of GARS, but not wild type, bind Neuropilin1 (NRP1) in vitro and antagonize its activity as a VEGF receptor in vivo. The motor nucleus of the facial nerve does not migrate caudally in GarsP278KY/+ embryos, resembling Nrp1 or Vegf knockouts. Nrp1 and GarsP278KY also interact genetically, and viral delivery of Vegf partially rescues the GarsP278KY/+ phenotype. However, questions remain, and mutant GARS may have additional pathogenic activities. Deletion of Nrp1 at P10 does not cause neuropathy, suggesting the mutant GARS/NRP1 interaction must occur earlier, and how much of the phenotype this mechanism explains and the cell autonomy of these interactions are unclear. To investigate these issues, in Aim 1A, we will look for developmental defects in Gars mutant mice that are consistent with NRP1 antagonism, and attempt to delete Nrp1 earlier to assess if this is sufficient to cause neuropathy. In Aim 1B, we will examine the effectiveness, timing, and cell autonomy of NRP1 decoy receptors in mitigating the activity of mutant GARS. Our second novel mechanism comes from work in Drosophila, where transgenic expression of disease-associated GARS alleles causes axon degeneration without altering tRNA charging, also suggesting a gain-of-function. However, global translation in affected neurons was reduced, and this was shown to be sufficient for axon degeneration. We will use an in vivo analysis of transcription and translation with cell-type-specific approaches including 4-thiouracil tagging of RNA, HA-epitope tagging of ribosomes, and noncanonical amino acid tagging of newly synthesized proteins to analyze RNA and proteins in the cell bodies and axons of peripheral neurons of control and mutant mice. We will determine if translation is suppressed in our Gars mutant mice, identify the specific RNA and proteins affected, determine whether this results from changes in RNA or translation, and whether the changes are specific to cell bodies or axons. This analysis is relevant to both gain- and loss-of- function mechanisms, and will improve our understanding of CMT2D and suggest new therapeutic strategies.